Non-invasive imaging of functional and molecular biomarkers in vivo is an emerging and important capacity in biological discovery, drug discovery and several clinical applications, which goes beyond anatomical imaging and retarded disease identification. Another important prospect of visualizing tissue biomarkers is the ability to examine and quantify treatment responses in vivo by monitoring specific primary molecules or downstream targets. Therapeutic efficacy could then be probed dynamically on timescales of hours to days. This ability is in contrast to the mainstay of today's healthcare with traditionally late end points of drug efficacy, a practice that often impairs prompt revision and exclusion of ineffective treatment strategies with potentially lethal results.
Similarly, while microscopy gives unprecedented insights into biology, it can only penetrate for a few hundred microns in tissues. Therefore the biological in vivo observation is limited by the microscopy penetration limit. Clearly methodologies that can penetrate deeper in tissue and visualize the microscopic contrast or utilize new contrast mechanisms are of immense importance in dynamic observations of biological phenomena, in developmental studies and in the drug discovery process.
Optical functional and molecular mesoscopic and macroscopic imaging of tissues has opened new pathways for study of many pathological processes in vivo. Indeed, optical wavelengths offer great variety of probing mechanisms that can be used for a variety of interrogations, from intrinsic functional information on blood oxygenation to molecular sensing. The use of extrinsically-administered fluorescent optical agents has further advanced the noninvasive photonic imaging by allowing visualization of otherwise invisible cellular and sub-cellular processes. For instance, the use of contrast agents and fluorescent reporters with specificity to proteins and enzymes has shown a high potential to differentiate several diverse disease biomarkers, such as inflammation and tumor progression.
U.S. Pat. No. 6,641,798 discloses tumor-targeted optical contrast agents useful for diagnostic imaging and therapy. The bioconjugates described include cyanine dyes with a variety of bis- and tetrakis (carboxylic acid) homologes. The compounds may be conjugated to bioactive peptides, carbohydrates, hormones, drugs, or other bioactive agents. The small size of the compounds allows more favorable delivery to tumor cells as compared to larger molecular weight imaging agents. These contrast agents are useful for diagnostic imaging and therapy, in endoscopic applications for the detection of tumors and other abnormalities, for localized therapy, for opto-acoustic tumor imaging, detection and therapy, and for sonofluorescence tumor imaging, detection and therapy. Fluorescence molecular tomography (FMT) is also capable of sensing picomole to femtomole quantities of fluorochromes in deep tissues at macroscopic scale, i.e., in whole animals with millimeter resolution. The technique shares tomographic principles with diffuse optical tomography and utilizes multi-projection illumination, combined with mathematical models that describe photon propagation in tissues, to reconstruct three-dimensional tomographic images of fluorochrome concentration.
U.S. Pat. No. 6,615,063 describes a fluorescence-mediated molecular tomographic imaging system, designed to detect near-infrared fluorescence activation in deep tissues. The system can use targeted fluorescent molecular probes or highly sensitive activable fluorescence molecular probes. Such probes add molecular specificity and yield high fluorescence contrast, to allow early detection and molecular target assessment of diseased tissue, such as cancers, in vivo.
Recently, tomographic imaging of tissues using opto-acoustics (photo-acoustics) has also demonstrated the ability to achieve penetration depths from several millimeters up to centimeters range with ultrasonic resolution. Opto-acoustic imaging relies on ultrasonic detection of opto-acoustically induced signals following absorption of pulsed light. The amplitude of the generated broadband ultrasound waves reflects local optical absorption properties of tissue. Since scattering of ultrasonic waves in biological tissues is extremely weak, as compared to that of light, biomedical opto-acoustic imaging combines high optical absorption contrast with good spatial resolution limited only by ultrasonic diffraction. Photo-acoustic imaging was proven efficient in imaging vascular trees, tumor angiogenesis, blood oxygenation monitoring, as well as sensitive to tissue chromophores, light-absorbing nanoparticles and dyes, and chromogenic assays.
For instance, U.S. Pat. No. 5,840,023 teaches a laser opto-acoustic imaging system, which utilizes time-resolved measurement of profiles of laser-induced transient pressure (acoustic) waves. The pressure waves are emitted by acoustic sources preferentially generated in absorbing tissues of diagnostic interest. This technique allows visualization of absorbed light distribution in turbid, layered and heterogeneous tissues irradiated by laser pulses in vivo. The laser opto-acoustic tomography can be used for the characterization of structure and properties of normal tissue, and for the detection of tissue pathological changes. The optical heterogeneities that can be imaged with the laser opto-acoustic imaging system include abnormal tissues such as tumors, injured tissues, blood vessels and other layered tissues. Further, three dimensional images of organs and portions of organs can be obtained.
Therefore, multi-spectral detection is often applied, as a means to better discriminate spectral signatures of various objects of interest. For example, U.S. Pat. No. 6,208,749 discloses a system for multi-spectral imaging of skin tissue that enables automatic characterization of the condition of a region of interest of the skin, based on direct digital imaging of that region or the digitization of its color photographic slides, when illuminating by appropriately filtered light. Parameters related to the texture, asymmetry, blotchiness and border irregularities are automatically estimated. The region of interest is automatically characterized by the digital processor, based on those parameters. The region of interest may include a skin lesion, in which case the characterization of the lesion as malignant or benign is enabled.
In U.S. Pat. No. 6,760,609, a method for determining an arterial blood oxygen saturation level by measuring the light transmittance through tissue of light of a first wavelength and a second wavelength is suggested. A steady-state component of the measured light transmission is used to select an appropriate calibration curve. A pulsatile component of the measured light transmission is used to determine the arterial blood oxygen saturation level using the selected calibration curves of oxy- and deoxy-hemoglobin spectral signatures. An oximetry system is further provided wherein a plurality of light transmission measurements are used to determine a blood oxygen saturation level.
In opto-acoustic spectroscopy, multi-wavelength methods were previously applied for differentiating blood chromophores (J. Laufer et al., “Phys. Med. Biol.” vol. 52, p. 141-168, 2007, U.S. Pat. No. 7,298,869).
U.S. Pat. No. 6,498,942 also discloses an opto-acoustic apparatus which includes a radiation source of pulsed radiation and a probe having a front face to be placed in close proximity to or in contact with a tissue site of an animal body. The probe further includes a plurality of optical fibers terminating at the surface of the front face of the probe and connected at their other end to a pulsed laser. The front face of the probe also has mounted therein or thereon a transducer for detecting an acoustic response from blood in the tissue site to the radiation pulses connected to a processing unit which converts the transducer signal into a measure of venous blood oxygenation. Another method, disclosed in US 2004/0127783, was suggested for imaging of dye markers by generating images with and without dye stimulation using two wavelengths (inside and outside the frequency band of fluorescence of the dye) and combining those for image enhancement.
A limitation of the above illumination techniques is that when operating with optically complex structures, such as tissue, the resulting images are a combined effect of the targeted chromophore and other native tissue chromophores. This complexity is particularly important in molecular imaging applications where molecular marker has to be resolved in the presence of many other non-specific tissue absorbers. In addition, opto-acoustic (or: photo-acoustic) observations so far have been limited to utilizing mono-directional homogenous illuminations, operating on the assumption that a similarly homogeneous illumination will occur as light propagates in tissue.
For example, WO 2007/084771 describes a method that delivers illumination which establishes “a homogeneous distribution of an energy fluence within any given plane or slice inside the body . . . .” Such illumination field is very difficult to achieve in practice, since tissue heterogeneity is not known and can impose significant variations of light intensity at any given plane inside tissue. When cylindrical objects are considered, such as the mouse torso, the conversion of mono-directional illumination in polar co-ordinates results in the utilization of multiple illumination points, arranges so that light is directed towards the center of the object, in the longitudinal sense. In this case, to simplify the illumination and detection arrangements, it is required that the tissue of investigation is surrounded by water or a similar fluid.
It could therefore be helpful to provide an improved imaging method, in particular for clinical and preclinical imaging or laboratory search purposes, which is capable of avoiding disadvantages of conventional techniques. In particular, it could be helpful to provide an imaging method which enables three-dimensional localization in tissues and quantification of molecular probes with increased precision. Furthermore, it could be helpful to provide an improved imaging device, in particular, being adapted for conducting the imaging method. The method and device are to be provided yielding, in particular, practical implementations and highly accurate discrimination of tissue biomarkers in vivo.